Abstract
Currently, the principal chemistries for the preparation of antibody–drug conjugates (ADC) target either lysines or cysteines for coupling cytotoxic drugs for delivery to target cells expressing tumor-specific antigens. All of these chemistries generate populations of molecules which differ in critical properties which are known to affect efficacy, pharmacokinetics, and the therapeutic window. Of key interest are methods to minimize this heterogeneity to achieve reproducible product profiles and efficacy. A current trend in the development of ADC is the evaluation of suitable targets, antibodies, and payloads, occurring well before process development to produce conjugates of clinical quality. This creates a need for an ability to generate comparably high-quality products early in development and at sufficient scale for evaluating in vitro potency and in vivo efficacy, as well as the early identification of any deficiencies in critical quality attributes including solubility and stability. Here we elaborate detailed protocols using maleimide-based chemistry for the conjugation to reduce hinge disulfides in antibodies by several cytotoxic drugs. We present a method for the initial characterization of the reduction/alkylation reaction using polyethylene-glycol (PEG) as a drug surrogate, a 5 mg scale drug conjugation to provide material for initial characterization including cell proliferation assays and a 150 mg scale process for performing efficacy studies in small animals. These methods yield well-defined predictable product profiles at high yield and with low impurities. These procedures include details relevant to the execution of these methods in a safe and contained manner within a typical laboratory environment.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Carter PJ, Senter PD (2008) Antibody-drug conjugates for cancer therapy. Cancer J 14:154–169
Senter PD (2009) Potent antibody drug conjugates for cancer therapy. Curr Opin Chem Biol 13:235–244
Younes A, Yasothan U, Kirkpatrick P (2012) Brentuximab vedotin. Nat Rev Drug Discov 11:19–20
Gualberto A (2012) Brentuximab Vedotin (SGN-35), an antibody-drug conjugate for the treatment of CD30-positive malignancies. Expert Opin Investig Drugs 21:205–216
Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF, Meyer DL, Francisco JA (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063–7070
Sun MM, Beam KS, Cerveny CG, Hamblett KJ, Blackmore RS, Torgov MY, Handley FG, Ihle NC, Senter PD, Alley SC (2005) Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug Chem 16:1282–1290
Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, Parsons-Reponte KL, Tien J, Yu SF, Mai E, Li D, Tibbitts J, Baudys J, Saad OM, Scales SJ, McDonald PJ, Hass PE, Eigenbrot C, Nguyen T, Solis WA, Fuji RN, Flagella KM, Patel D, Spencer SD, Khawli LA, Ebens A, Wong WL, Vandlen R, Kaur S, Sliwkowski MX, Scheller RH, Polakis P, Junutula JR (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol 30:184–189
Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, Bhakta S, Nguyen T, Dugger DL, Li G, Mai E, Lewis Phillips GD, Hiraragi H, Fuji RN, Tibbitts J, Vandlen R, Spencer SD, Scheller RH, Polakis P, Sliwkowski MX (2010) Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res 16:4769–4778
Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925–932
Kovtun YV, Audette CA, Ye Y, Xie H, Ruberti MF, Phinney SJ, Leece BA, Chittenden T, Blattler WA, Goldmacher VS (2006) Antibody-drug conjugates designed to eradicate tumors with homogeneous and heterogeneous expression of the target antigen. Cancer Res 66:3214–3221
Okeley NM, Miyamoto JB, Zhang X, Sanderson RJ, Benjamin DR, Sievers EL, Senter PD, Alley SC (2010) Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res 16:888–897
Pollack VA, Alvarez E, Tse KF, Torgov MY, Xie S, Shenoy SG, MacDougall JR, Arrol S, Zhong H, Gerwien RW, Hahne WF, Senter PD, Jeffers ME, Lichenstein HS, LaRochelle WJ (2007) Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB. Cancer Chemother Pharmacol 60:423–435
Luo S, Wehr NB, Levine RL (2006) Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green. Anal Biochem 350:233–238
Spack EG Jr, Packard B, Wier ML, Edidin M (1986) Hydrophobic adsorption chromatography to reduce nonspecific staining by rhodamine-labeled antibodies. Anal Biochem 158:233–237
Ejima D, Yumioka R, Arakawa T, Tsumoto K (2005) Arginine as an effective additive in gel permeation chromatography. J Chromatogr A 1094:49–55
Ricker RD, Sandoval LA (1996) Fast, reproducible size-exclusion chromatography of biological macromolecules. J Chromatogr A 743:43–50
Yumioka R, Sato H, Tomizawa H, Yamasaki Y, Ejima D (2010) Mobile phase containing arginine provides more reliable SEC condition for aggregation analysis. J Pharm Sci 99:618–620
Rehder DS, Dillon TM, Pipes GD, Bondarenko PV (2006) Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics. J Chromatogr A 1102:164–175
Dillon TM, Bondarenko PV, Rehder DS, Pipes GD, Kleemann GR, Ricci MS (2006) Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. J Chromatogr A 1120:112–120
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Stefano, J.E., Busch, M., Hou, L., Park, A., Gianolio, D.A. (2013). Micro- and Mid-Scale Maleimide-Based Conjugation of Cytotoxic Drugs to Antibody Hinge Region Thiols for Tumor Targeting. In: Ducry, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 1045. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-541-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-62703-541-5_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-540-8
Online ISBN: 978-1-62703-541-5
eBook Packages: Springer Protocols